Single Atom‐Doped Nanosonosensitizers for Mutually Optimized Sono/Chemo‐Nanodynamic Therapy of Triple Negative Breast Cancer

Abstract Sonodynamic therapy (SDT) represents a promising therapeutic modality for treating breast cancer, which relies on the generation of abundant reactive oxygen species (ROS) to induce oxidative stress damage. However, mutant breast cancers, especially triple‐negative breast cancer (TNBC), have evolved to acquire specific antioxidant defense functions, significantly limiting the killing efficiency of SDT. Herein, the authors have engineered a distinct single copper atom‐doped titanium dioxide (Cu/TiO2) nanosonosensitizer with highly catalytic and sonosensitive activities for synergistic chemodynamic and sonodynamic treatment of TNBC. The single‐atom Cu is anchored on the most stable Ti vacancies of hollow TiO2 sonosensitizers, which not only substantially improved the catalytic activity of Cu‐mediated Fenton‐like reaction, but also considerably augmented the sonodynamic efficiency of TiO2 by facilitating the separation of electrons (e−) and holes (h+). Both the in vitro and in vivo studies demonstrate that the engineered single atom‐doped nanosonosensitizers effectively achieved the significantly inhibitory effect of TNBC, providing a therapeutic paradigm for non‐invasive and safe tumor elimination through the mutual process of sono/chemo‐nanodynamic therapy based on multifunctional single‐atom nanosonosensitizers.

(BioTek Instruments, United States). ESR experiment was conducted on a JEOL-FA200 spectrometer. Confocal laser scanning microscopy images (CLSM) were acquired by FV1000 Olympus Co. US irradiation for sonodynamic anti-tumor therapy was performed by an Intelligent Transport Ultrasound (Chattanooga Group, USA).

Synthesis and Surface NH 2 -mPEG 2000 Modification of Cu/TiO 2 Nanoparticles (Cu/TiO 2 -PEG):
The single-atom Cu/TiO 2 nanoparticles were synthesized according to the published literature. [1] Firstly, spherical silica (SiO 2 ) nanoparticles were synthesized under alkaline conditions. The TEOS (0.86 mL) was dissolved in a mixed solution, which contained H 2 O (4.3 mL), ammonia (0.6 mL), and ethanol (23 mL). Then, the mixed solution was stirred at room temperature for 6 hours. The SiO 2 nanoparticles were collected by centrifugation and dispersing in ethanol (40 mL). Secondly, ammonia (0.4 mL, 28 -30 wt%) and pure acetonitrile (14 mL) were mixed with the above SiO 2 nanoparticles in solution and stirred for 30 min (called solution a). Then, TBOT (0.8 mL) was added to acetonitrile (2 mL) and 6 mL absolute ethanol solution (called solution b). Solution b was slowly dripped to solution a and kept stirring for 3 h to cover with TiO 2 . The products were centrifuged and washed with H 2 O several times to obtain SiO 2 @TiO 2 nanoparticles and dispersed in H 2 O (40 mL). Thirdly, CuCl 2 ·2H 2 O (1 mL, 6 mg/mL) solution was added to the previously SiO 2 @TiO 2 nanoparticles and stirred uniformly for several hours. After stirring, the SiO 2 @CuO x /TiO 2 nanoparticles were obtained by centrifugation and washing with H 2 O and dispersed in H 2 O (40 mL).
Fourthly, PVP (400 mg) was dispersed in H 2 O (20 mL) to obtain an aqueous PVP solution, which was added to SiO 2 @CuO x /TiO 2 solution. After the addition of PVP, the mixture was stirred for 8 h to make the adsorption of PVP onto SiO 2 @CuO x /TiO 2 nanoparticles. After the adsorption of PVP, the products were separated by centrifugation and scattered in H 2 O (8.6 mL) and ethanol (46 mL) solution. Then TEOS (1.6 mL) and ammonia (1.2 mL, 28 -30 wt%) were added to the above solution, then formed SiO 2 coating (SiO 2 @CuO x /TiO 2 @SiO 2 nanoparticles). After 4 h, the products were centrifuged and dried overnight in a lowtemperature dryer and grind in mortar for uniformity. Fifthly, to spatially redistribute Cu single-atoms, the dried nanoparticles were calcined at 900 °C for 2 h to yield SiO 2 @Cu/TiO 2 @SiO 2 nanoparticles. Finally, the nanoparticles were dispersed in NaOH solution (20 mg mL -1 ) and heated to 90 ℃ by uniformly stirring. After 6 h, the products were centrifuged and washed with H 2 O to yield an aqueous Cu/TiO 2 solution. The products were frozen overnight at a low temperature and used for characterization further use.
For enhancing the stability and biosafety of Cu/TiO 2 nanoparticles under physiological conditions, biocompatible methoxypolyethylene glycol amine 2000 (NH 2 -mPEG 2000 ) was grafted onto the surface of Cu/TiO 2 nanoparticles. 5 mL of Cu/TiO 2 nanoparticles solution ([Ti]: 400 ppm) and NH 2 -mPEG 2000 (40 mg) were added into a necked bottle, which was sonicated for 60 min and then stirred overnight. The Cu/TiO 2 -PEG was collected by centrifugation and washed several times with H 2 O to remove the non-attached PEG.
In Vitro ROS Generation of Cu/TiO 2 by US Activation: 3, 3', 5, 5'-tetramethylbenzidine (TMB) was used to verify the occurrence of the Fenton reaction, based on the production of ·OH. Cu/TiO 2 (100 μL, 300 ppm) was mixed with TMB in acidic solution (0.01 mM TMB testing solution 100 μL, 100 μM H 2 O 2 solution 100 mL, PBS buffer 1.7 mL, pH 6.5). The absorbance change of the TMB working solution at 652 nm was recorded every minute, for a total of 20 minutes. Similar operations were performed for the other groups (control and TiO 2 ).
Meanwhile, the absorbance changes of the TMB working solution with or without US irradiation (power density: 1.0 W cm -2 , duty cycle: 50 %, 5 min) were recorded after 20 minutes under different concentrations of Cu/TiO 2 (0,50,100,150,200,250, and 300 ppm).
A singlet oxygen probe, 1,3-diphenylisobenzofuran (DPBF), was employed for characterizing the effects of US-activated Cu/TiO 2 nanosonosensitizers on ROS-generating efficacy in vitro. In a typical assay, DPBF (2 mg mL -1 , 40 μL) and Cu/TiO 2 nanoparticles (100 μL, Ti concentration: 200 ppm) were mixed with DMF (2.86 mL). Then, the mixed solution was subjected to irradiation by US (50 % duty cycle, 1 MHz, 1.0 w cm -2 ) for various lengths of time in the dark (0, 2.5, 5, 7.5, 10, and 12.5 min). The variation in absorbance of DPBF at 416 nm was identified using the UV-vis-NIR spectrum. Meanwhile, the ROS generation of different concentrations of Cu/TiO 2 was verified under US activation (0, 50, 100, 150, 200, and 250 ppm). Package (VASP). [3] Taking into account the influence of Van der Waals interactions, DFT-D3 function was employed. [4] The electronic plane wave interception energy was set to 350 eV, and these configurations were optimized until the energy differences were converged within 10 -4 eV and the forces of all atoms were less than 0.01 eV/Å. The vacuum layers between neighboring images were set to be larger than 15 Å, which was enough to avoid the interactions between neighboring images. The models were constructed of 2×2×5 TiO 2 unit cells with periodic boundary conditions (PBC) in plane and the bottom two layers were fixed to simulate the semi-infinite substrate bulk. The 5×5×1 k-point Gamma meshes were performed for these models.

Electron Spin Resonance (ESR) Spectra Test in Vitro
Cell Culture: The Cell Bank of the Chinese Academy of Sciences in Shanghai provided the 4T1 mouse breast cancer cell line. The 4T1 Cells were grown in DMEM with 10% foetal bovine serum (FBS), 1% penicillin/streptomycin, and 5% CO 2 humidified conditions at 37 °C.
In Vitro Cytotoxicity Assay: For the in vitro cytotoxicity tests, 4T1 cells were employed. 96well plates were selected for culturing 4T1 cells at 1×10 4 cells per well, in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, and a humidifier controlled at 5% CO 2 and 37 °C. To evaluate the cytotoxicity of synergistic single-atom catalysis and SDT using the CCK-8 assays, 4T1 cells were cultured with 100 μL of DMEM containing different concentrations of Cu/TiO 2 -PEG ([Ti]: 0, 6, 12, 25, 50, 100 and 200 ppm) in 96 well plates and were irradiated with the US after 4 h of co-incubation. Then, cell viabilities were then subjected to a standard CCK-8 assay after 24 h. After the solution was washed by PBS thrice, 100 μL of DMEM containing 10 μL of CCK8 solution was added to each well, and coincubated for 1 h, which was measured in a SynergyHTX microplate reader at 450 nm. US irradiation conditions were adjusted as 50 % duty cycle, 1.0 MHz, and 1.0 W cm -2 , 5 min.

Endocytosis Analysis of Cu/TiO 2 -PEG as Observed via Flow Cytometry and CLSM:
To track the endocytosis of Cu/TiO 2 -PEG, CLSM was employed. Seeding of 1× 10 5 4T1 cells was made into dishes made specifically for CLSM, and the dishes were then cultured in a humid incubator. Then, 1 ml of DMEM containing FITC-labeled Cu/TiO 2 -PEG nanosonosensitizers was added in place of the original media. After different incubation durations (0, 2, 4, and 8 h), CLSM was carried out after staining with DAPI for 15 min and washing with PBS.
To further perform the endocytosis of Cu/TiO 2 -PEG nanosonosensitizers by the flow cytometry, 6-well plates were used to plate 4T1 cells at 3 × 10 5 cells per well and 2 ml of DMEM supplemented with FITC-labeled Cu/TiO 2 -PEG nanosonosensitizers was used to replace the medium for various incubation periods (0, 2, 4 and 8 h). Specific test tubes were used to collect 4T1 cells, and the average fluorescence intensities of FITC-labeled Cu/TiO 2 -PEG in 4T1 cells were analyzed by flow cytometry.

In Vitro Synergistic Single-atom Catalysis and SDT Effects as Observed by Flow Cytometry
and CLSM: In a dish designed for CLSM, 4T1 cancer cells were plated and set to co-incubate with the appropriate Cu/TiO 2 -PEG nanosonosensitizers. The cells underwent a 24-hour treatment period using a variety of procedures, including the control group, the only US group, the TiO 2 -PEG group, the TiO 2 -PEG + US group, the Cu/TiO 2 -PEG group, and the Cu/TiO 2 -PEG + US group. For 5 min, the cells were treated with US (50% duty cycle, 1 MHz, and 1.0 W cm -2 ). The media was then taken out and stained for 15 minutes with calcein-AM and PI solution. The monitoring of the cells was completed by CLSM, in which the dead cells were labeled with red and the living cells with green.
4T1 cells were cultured in a 6-well plate and grew to a percentage of 70 to 80% in order to analyze apoptosis using flow cytometry. As previously noted, cells were subjected to treatments after being cultivated for 24 hours. With 500 μL of binding buffer, cells were collected and disseminated once more. FITC (15 μL) and Annexin V (5 μL) were utilized for labeling the living and dead cells for a duration of 20 min. Flow cytometry was ultimately used to identify cell apoptosis.
In Vitro Generation of ROS as Performed by CLSM and Flow Cytometry: In a CLSM-specific culture dish, 4T1 cells were seeded (1 × 10 5 cells per dish) and then distributed into six groups: control group, only US group, TiO 2 -PEG group, TiO 2 -PEG + US group, Cu/TiO 2 -PEG, and Cu/TiO 2 -PEG + US group. The Cu/TiO 2 -PEG nanosonosensitizers were allowed to incubate with cells for a period of 4 h. Cells were subsequently treated with US (50 % duty cycle, 1 MHz, and 1.0 W cm -2 ) for 5 min. Appropriate amounts of DCFH-DA were added to individual culture dishes for 20 min. Finally, CLSM was used for observing the cells.
The generation of ROS in 4T1 cells was detected using flow cytometry. Typically, 6-well culture plates were used to culture 4T1 cells (3 × 10 5 cells per well). The cells, which were treated as mentioned above, were collected and the fluorescence intensity of DCFH-DA was detected following centrifugation. The images were taken at 0, 1,2,4,8,12, and 24 h following injection. Subsequently, mice were euthanized and tumor nodules and key organs (heart, lung, kidney, spleen, and liver) were gathered for analysis of ex vivo FL intensities.

Analysis of Gene
In Vivo Biocompatibility Evaluation: Random division of twenty female ICR mice resulted in four groups and assessed for the in vivo biocompatibility and biosafety studies (n = 5 per group), including (1) control (intravenous injection with 100 μL of saline), (2) 1.25 mg kg −1 (intravenous injection with 100 μL of Cu/TiO 2 -PEG at a dose of 1.25 mg kg −1 ), (3) 2.5 mg kg −1 (intravenous injection with 100 μL of Cu/TiO 2 -PEG at a dose of 2.5 mg kg −1 ), and (4) 5 mg kg −1 (intravenous injection with 100 μL of Cu/TiO 2 -PEG at a dose of 5 mg kg −1 ). After feeding for 30 days, twenty mice were euthanized, and blood specimens and main organs (heart, lung, kidney, spleen, and liver) were utilized for blood investigations and histopathological analysis, respectively.

In Vivo Synergistic Single-atom Catalysis and SDT of Cu/TiO 2 -PEG under US Irradiation:
For in vivo synergistic single-atom catalysis and SDT therapy, 4T1 cells suspended in 100 μL PBS were injected subcutaneously into the right back (1 × 10 6 cells/site) of the female BALB/C nude mice. Random division of the mice was made into six groups following the growth of the tumor to a mean volume of about 70 mm 3  Two-way ANOVA and Student's two-tailed t-test were employed for determining the statistical significance of the comparison among the two groups (*P< 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).                   Figure S19. H&E staining of the major organs (heart, liver, spleen, lung and kidney) of 4T1 tumor-bearing mice after various treatments. Scale bar: 100 µm.